Dynamics of Phosphorylation and Assembly of the High Molecular Weight Neurofilament Subunit in NB2a/d1 Neuroblastoma

: In neuronal systems thus far studied, newly synthesized neurofilament subunits rapidly associate with the Triton‐insoluble cytoskeleton and subsequently undergo extensive phosphorylation. However, in the present study we demonstrate by biochemical and immunological criteria that NB2a/dl neuroblastoma cells also contain Triton‐soluble, extensively phosphorylated 200‐kDa high molecular weight neurofilament subunits (NF‐H). High‐speed centrifugation (100,000 g) of the Triton‐soluble fraction for 1 h sedimented some, but not all, soluble NF‐H subunits; immunoelectron microscopic analyses of the resulting pellet indicated that a portion of the NF‐H subunits in this fraction are assembled into (Triton‐soluble) neurofilaments. When cells were pulse labeled for 15 min with [35S]methionine, radiolabel was first associated with the Triton‐soluble 200‐kDa NF‐H variants. Because only extensively phosphorylated NF‐H subunits migrate at 200 kDa, whereas hypophosphorylated subunits migrate instead at 160 kDa, these findings suggest that some newly synthesized subunits were phosphorylated before they polymerized. In pulse‐chase analyses, radiolabeled 200‐kDa NF‐H migrated into the 100,000 g particulate fraction of Triton‐soluble extracts before its arrival in the Triton‐insoluble cytoskeleton. Undifferentiated cells, which do not possess axonal neurites and lack a significant amount of Triton‐insoluble, extensively phosphorylated NF‐H, contain a sizeable pool of Triton‐soluble extensively phosphorylated NF‐H subunits and polymers. We interpret these data to indicate that the integration of newly synthesized NF‐H into the cytoskeleton occurs in a progression of distinct stages, and that assembly of NF‐H into neurofilaments and integration into the Triton‐insoluble cytoskeleton are not prerequisites for the incorporation of certain phosphate groups on these polypeptides. Because NF‐H can be extensively phosphorylated in perikarya, additional mechanisms besides differential localization of the responsible kinase systems must account for the segregation of Triton‐insoluble NF‐H in NB2a/d1 neurites.